Pharmaceutical use of α antigen or α antigen gene

ABSTRACT

The α antigen-encoding gene and the α antigen protein suppress the production of interleukin-4 etc., improve the Th2 type cytokine-dominant state, and furthermore inhibit various conditions of allergic diseases such as IgE production, histamine release and eosinophil infiltration, and therefore they are very effective for the prevention or treatment of atopic diseases such as atopic dermatitis, asthma, allergic rhinitis, and allergic conjunctivitis, and more broadly allergic diseases.

This is a continuation of application Ser. No. 11/352,342 filed Feb. 13,2006 now U.S. Pat. No. 7,524,675, which is a divisional of U.S.application Ser. No. 10/468,456, filed Aug. 20, 2003, now abandoned,which is a 371 of PCT/JP02/01459, filed Feb. 20, 2002. The entiredisclosures of each of the prior applications are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to novel pharmaceutical uses of α antigenderived from acid-fast bacteria (Mycobacteria) or analogs thereof orgenes encoding them. More specifically, it relates to novelpharmaceutical uses of α antigen derived from Mycobacterium kansasii oranalogs thereof or expression vectors containing a gene encoding themfor the prevention or treatment of allergic diseases such as atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis.

BACKGROUND ART

Allergic diseases such as atopic dermatitis, asthma, allergic rhinitisand allergic conjunctivitis are diseases in which hypersensitivereactions occur against environmental antigens to which normal healthypeople do not react and destruction and disorders of various organsoccur due to the autoimmune system. As an onset mechanism of thesediseases, there has been considered the enhanced allergic reactionscaused by Th2 type cytokines such as interleukin-4 and interleukin-5that the Th2 cells among the Th differentiate involved in cellularimmune responses against antigen (Progress in Medicine 17: 19-20, 1997).The elucidation of the induction mechanism and the control mechanism hasimportant physiological and pharmacological implications, but detailedmechanisms thereof have yet to be clarified. Therapies of these diseasesin current use include evasion from antigen, the control of non-specificinflammatory reactions by the oral administration of antihistamines thatantagonize the binding of mediators such as histamine to receptors andby topical steroid (Igaku No Ayumi (Journal of Clinical and ExperimentalMedicine) 180: 51-55, 1997).

For the treatment of allergic diseases, there can be conceived thesuppression of allergic reactions by shifting the Th2 typecytokine-dominant allergic state to the Th1 type cytokine-dominantstate, and since interferon-γ produced by Th1cells suppresses the effectof enhancing IgE production by interleukin-4 produced by Th2 cells(Progress in Medicine 17: 19-20, 1997), interferon-γ has been in trialuse for the treatment of allergic diseases (J. Am. Acad. Dermatol. 32:684-685, 1991; Allergy 49: 120-128, 1994; Acta Derm. Venereol 73:130-132, 1993), but the effect is small and the results have not beensatisfactory, and thus has not been subjected to clinical uses.

For the establishment of atopic diseases, the maintenance of the Th2type-dominant immunological state and the maintenance of ensuinginflammatory reactions are involved. For the improvement of the Th2type-cytokine dominant immunological state, control of cytokines byimmunosuppressive agents has been attempted (Br. J. Dermatol. 143:365-72, 2000; J. Allergy Clin. Immunol. 106 (1 pt2): S58-64, 2000), butthat did not lead to the essential improvement of the immunologicalstate and thereby had a limited effect.

On the other hand, BCG vaccine is a vaccine that utilizes an attenuatedstrain of Mycobacterium bovis and is the only live vaccine approved forMycobacterium tuberculosis infections. BCG vaccine has a potent adjuvanteffect with little side effects, and thus has been given to many peoplein the world today as a safe vaccine. Now, BCG vaccine has been reportedto have an activity of shifting CD4+ helper T cells to Th1 cells thatare responsible for cell-mediated immunity by producing interferon-γ andinterleukin-2 (Cancer Immunol. Immunother. 39: 401-406, 1994; ibid. 40:103-108, 1995).

As a protein that is omnipresent among Mycobacteria, the antigen 85complex was identified. The protein complex is composed of an antigen 85complex-forming protein 85A with a molecular weight of about 30-32 kd(Infect. Immun. 57:3123-3130, 1989), an antigen 85 complex-formingprotein 85B (J. Bacteriol. 170: 3847-3854, 1988) and an antigen 85complex-forming protein 85C (Infect. Immun. 59: 3205-3212, 1991), whichare major secretory proteins of Mycobacteria. These secretory proteinsexhibit high homology of the gene sequence and the amino acid sequenceand cross reactivity to monoclonal antibodies among the bacteria of thesame genera such as Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium kansasii etc. irrespective of the species (Microbiol. Rev.56: 648-661, 1992) and have, as common functions, the activity ofbinding to fibronectin and of mycolyl group transferase in the cell wallsynthesis (Microbiol. Rev. 56: 648-661, 1992; Science 276: 1420-1422,1997; Nat. Struct. Biol. 7: 887-88, 2000). Among these secretoryproteins, the antigen 85 complex-forming protein 85B is widely known asα antigen.

Currently, furthermore, α antigen has been isolated and purified as atuberculin reactive protein from the culture supernatant ofMycobacterium tuberculosis, and it has been revealed, there is anepitope in the molecule (Am. Rev. Respir. Dis. 130: 647-649, 1984; ibid.132: 173-174, 1985; Microbiological Reviews 56: 648-661, 1992), and ithas been reported that this α antigen has the above-mentioned effect ofshifting to the Th1 cells (Infect. Immun. 60: 2880-2886, 1992). Attemptshave also been made to use and improve Bacillus Calmette-Guerin byrecombinant DNA technology, and then to use as a vaccine to variouspathogens. For example, it has been reported, an expression vector wasconstructed in which a gene encoding the surface antigen of AIDS viruswas integrated into the gene containing the α antigen, and the vectorwas used to transform Bacillus Calmette-Guerin, which transformant isused as a BCG vaccine (WO 96/4009).

Up until today, however, no attempts have been made to use a geneencoding the α antigen or the α antigen per se for the treatment ofallergic diseases. Furthermore, though the α antigen has been reportedto have the effect of shifting CD4+ helper T cells to Th1 cells, it isnot yet clear whether the α antigen or the α antigen gene is effectivefor the prevention or treatment of allergic diseases such as atopicdermatitis and asthma for which the mechanism of onset has not beenelucidated. In addition, as described above, though the shifting of theTh1 type/Th2 type balance to the Th2 type-dominant state is consideredto be important for the establishment of allergic diseases, no reportshave been made so far that the mere shifting to the Th1 cell side led tothe improvement of skin conditions of atopic dermatitis.

DISCLOSURE OF THE INVENTION

Thus, it is an object of the present invention to provide novelpharmaceutical use of the Mycobacterium-derived α antigen such as BCGbacteria or analogs thereof or genes encoding them for the prevention ortreatment of allergic diseases.

The present inventors have found that when an expression vectorcontaining the α antigen-coding gene is applied to Caspase-1 transgenicmice, a mouse model which is at the Th2 type cytokine-dominantimmunological state and which has a persistent atopic dermatitis-likedermatitis, the production of interleukin-4 was inhibited, blood levelsof histamine and IgE were also suppressed, and skin diseases wereimproved, indicating that atopic dermatitis can be healed. The α antigenprotein was also found to heal atopic dermatitis in a similar manner.Furthermore, when an expression vector containing the α antigen-codinggene was applied to a mouse asthma model, it was found, IgE productionwas inhibited and allergic conditions such as eosinophil-infiltrationcan be improved, leading to the healing of asthma. Thus, the presentinventors have revealed that the α antigen gene or the α antigen proteinimproves the Th2 type cytokine-dominant immunological state and cansuppress and/or improve various conditions of allergic diseases, andthus is widely effective for the prevention or treatment of allergicdiseases, and thereby have completed the present invention.

Thus, the present invention relates to a pharmaceutical composition forthe prevention or treatment of allergic diseases comprising, as anactive ingredient, the Mycobacterium-derived α antigen, an analogthereof, a mutant thereof having a function similar thereto, or a geneencoding them.

Furthermore, the present invention relates to a method of preventing ortreating allergic diseases, said method comprising administering aneffective amount of the Mycobacterium-derived α antigen, an analogthereof, a mutant thereof having a function similar thereto, or a geneencoding them to mammals including humans.

Furthermore, the present invention relates to the use of theMycobacterium-derived α antigen, an analog thereof, a mutant thereofhaving a function similar thereto, or a gene encoding them for theproduction of a pharmaceutical composition for the prevention ortreatment of allergic diseases.

According to a preferred embodiment, the present invention uses anexpression vector encoding the Mycobacterium-derived α antigen or ananalog thereof, or the α antigen protein or an analog protein thereoffor the prevention or treatment of atopic diseases such as atopicdermatitis, asthma, allergic rhinitis, and allergic conjunctivitis. Asused herein, as analogs of the α antigen, there can be mentioned anantigen 85 complex-forming protein 85A, antigen 85 complex-formingprotein 85C and the like.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows the construction of an expression vector, constructed inExample 1, containing a gene encoding the α antigen for use as an activeingredient of a pharmaceutical composition for the prevention ortreatment of allergic diseases of the present invention.

FIG. 2 is a drawing showing the effect of an expression vectorcontaining a gene encoding the α antigen on the treatment of atopicdermatitis.

FIG. 3 is a graph showing serum levels of IgE when an expression vectorcontaining a gene encoding the α antigen was administered to a mouseasthma model.

FIG. 4 is a graph showing protein concentrations in the alveolar lavagewhen an expression vector containing a gene encoding the α antigen wasadministered to a mouse asthma model.

FIG. 5 is a graph showing eosinophil counts in the alveolar lavage whenan expression vector containing a gene encoding the α antigen wasadministered to a mouse asthma model.

FIG. 6 is a graph showing the degree of infiltration of eosinophils inthe lung tissue when an expression vector containing a gene encoding theα antigen was administered to a mouse asthma model.

FIG. 7 is a graph showing the result of histological examination of thelung when an expression vector containing the gene encoding the αantigen was administered to a mouse asthma model.

BEST MODE FOR CARRYING OUT THE INVENTION

The subject diseases of the present invention are allergic diseases, andmore specifically allergic diseases caused by the Th2 typecytokine-dominant state. The preferred subject allergic diseases of thepresent invention are specifically atopic diseases, for example atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis, andspecifically atopic dermatitis and asthma.

As used herein, genes encoding the Mycobacterium-derived α antigen or ananalog thereof refers to genes capable of expressing the α antigenprotein, or α antigen protein analogs such as antigen 85 complex-formingprotein 85A and antigen 85 complex-forming protein 85C. Specifically,there can be mentioned genes in the form of an expression vectorcontaining a gene encoding the α antigen or an analog thereof. As genesencoding the α antigen, there can be illustrated genes encoding the αantigen derived from Mycobacteria such as Mycobacterium kansasii(Infect. Immun. 58: 550-556, 1990), Mycobacterium avium (Infect. Immun.61: 1173-1179, 1993), Mycobacterium intracellulare (Biochem. Biophys.Res. Commun. 196: 1466-1473, 1993), Mycobacterium leprae (Mol.Microbiol. 6: 153-163, 1992) and the like. Any of these genes can beused in the present invention, and as a gene encoding α antigen derivedfrom Mycobacterium kansasii, there can be mentioned a DNA having thebase sequence from positions 390 to 1244 of SEQ ID NO: 1.

In addition to this DNA, they may be a mutant DNA that hybridizes tothis DNA under a stringent condition, or a mutant DNA comprising a DNAencoding a protein having an amino acid sequence in which one or morethan one (preferably several) amino acid residues have been substituted,deleted and/or added to the amino acid sequence of the protein encodedby this DNA, wherein said mutant encodes a protein having the samefunction as the Mycobacterium kansasii-derived α antigen. As usedherein, as a specific method of obtaining a mutant DNA that hybridizesto this DNA under a stringent condition, there can be mentioned thefollowing method. Thus, a colony hybridization is performed in thepresence of 50% formamide, 4×Denhardt, 5×SSPE (SSPE solution: EDTAsodium phosphate (SSPE), 1×Denhardt: 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.2% SDS, 100 μg/ml ssDNA, and12.5 ng of a probe (12.5 ng of a purified cDNA fragment having a basesequence from positions 390 to 1244 of SEQ ID NO: 1 labelled with[α-³²P]dCTP (Amersham) using the BcaBest DNA Labeling kit (TaKaRa)) at45° C. for 14-16 hours, the filter is washed in 1×SSPE and a 0.5% SDSsolution at 45° C./30 min, then in 0.1×SSPE and a 0.5% SDS solution at55° C./1 hour, and finally 0.1×SSPE and a 0.5% SDS solution at 65° C./1hour to eliminate the background completely, which is then exposed to anX-ray film (Fuji) at −80° C. for 72 hours to determine the position ofand isolate the corresponding colony and thus the mutant DNA can beobtained. The above same function as the α antigen derived fromMycobacterium kansasii means to have a similar effect of preventing ortreating allergic diseases. These mutants are those for which amino acidsequences encoded thereby usually have a homology of 60% or greater withthe amino acid sequence of the α antigen protein, and preferably ahomology of 75% or greater. In the case of genes encoding the α antigenother than Mycobacterium kansasii, they may be mutants thereof as well.

As genes encoding the antigen 85 complex-forming protein 85A which is ananalog of the α antigen, there can be mentioned genes derived from theMycobacteria similar to various Mycobacteria described above for the αantigen gene. More specifically, there can be mentioned a DNA encodingthe antigen 85 complex-forming protein 85A derived from Mycobacteriumtuberculosis (Infect. Immun. 57: 3123-3130, 1989). For genes encodingthe antigen 85 complex-forming protein 85C as well, there can bementioned a gene derived from various Mycobacteria, and morespecifically, there can be mentioned a DNA encoding the antigen 85complex-forming protein 85C derived from Mycobacterium tuberculosis(Infect. Immun. 59: 3205-3212, 1991). For these DNAs as well, asdescribed antibody, they may be a mutant DNA that hybridizes to this DNAunder a stringent condition, or a mutant DNA comprising a DNA encoding aprotein having an amino acid sequence in which one or more than one(preferably several) amino acid residues have been substituted, deletedand/or added to the amino acid sequence of the protein encoded by thisDNA, wherein said mutant encodes a protein having the same function asthe antigen 85 complex-forming protein 85A or the antigen 85complex-forming protein 85C.

The above DNAs can be cloned by a methyl such as RT-PCR reaction on mRNAderived from Mycobacteria using as PCR primers appropriate DNA segmentsbased on the sequence information described in the above literature,sequence information of Genebank, etc. Chemical synthesis is alsopossible based on the amino acid sequence information. Furthermore, theabove DNA mutants may be easily obtained using, for example,site-directed mutagenesis, PCR method, a common hybridization method, orthe like.

In accordance with the present invention, the α antigen protein derivedfrom Mycobacteria or derivatives thereof such as antigen 85complex-forming protein 85A or antigen 85 complex-forming protein 85C ormutant proteins thereof can be used for the prevention or treatment ofallergic diseases. As such α antigens, there can be mentioned proteinsencoded by genes encoding the above-mentioned α antigen. Specifically,there can be mentioned an α antigen which is encoded by the DNA havingthe base sequence derived from Mycobacterium kansasii described in SEQID NO: 1 and which has the amino acid sequence of SEQ ID NO: 2. Inaddition to the α antigen having this amino acid sequence, it may be amutant protein comprising an amino acid sequence in which one or morethan one (preferably several) amino acid residues have been substituted,deleted and/or added to the amino acid sequence of SEQ ID NO: 2, saidprotein having the same function as the α antigen. In the case of the αantigen derived from Mycobacterium other than Mycobacterium kansasii aswell, it may be a mutant protein comprising a protein in which one ormore than one (preferably several) amino acid residues have beensubstituted, deleted and/or added to the amino acid sequence of thoseamino acid sequences, said protein having the same function as the αantigen. For the antigen 85 complex-forming protein 85A or the antigen85 complex-forming protein 85C as well, there can be mentioned theantigen 85 complex-forming protein 85A derived from Mycobacteriumtuberculosis (Infect. Immun. 57: 3123-3130, 1989), the antigen 85complex-forming protein 85C derived from Mycobacterium tuberculosis(Infect. Immun. 59: 3205-3212, 1991) and the like. The analogs of theseα antigen proteins may be mutants similar to those of the α antigenprotein mentioned above.

These proteins may be produced by a recombinant DNA technology usinggenes encoding them or by chemical synthesis. Alternatively,Mycobacteria such as Mycobacterium kansasii may be cultured in asuitable medium, and from the culture liquid, the proteins may bepurified by a known purification method (Scand. J. Immunol. 43: 202-209,1996; J. Bacteriol. 170: 3847-3854, 1988; Hiroshima J. Med. Sci. 32:1-8, 1983).

In accordance with the present invention, when genes encoding theMycobacterium-derived α antigen, a derivative thereof, or a mutantthereof are used for the prevention or treatment of allergic diseases,specifically they may be used in the form of an expression vectorcontaining a gene encoding the α antigen, an analog thereof, or a mutantthereof. Expression vectors containing these genes are roughly dividedinto two: cases when non-virus vectors are used, and cases when virusvectors are used.

Non-virus vectors may be any expression vectors as long as they canexpress and secrete genes that encode the α antigen, a derivativethereof, or a mutant thereof, and by way of example, there can bementioned pCAGGS (Gene 108: 193-200, 1991), pBK-CMV, pcDNA3.1, pZeoSV(Invtrigen, Stratagene) etc.

As virus vectors, representative virus vectors include recombinantadenovirus, retrovirus and the like. More specifically, there can bementioned DNA viruses or RNA viruses such as detoxicated retrovirus,adenovirus, adeno-associated virus, helper virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, and humanimmunodeficiency virus (HIV).

By integrating a gene encoding the α antigen, an analog thereof or amutant thereof into these vectors, expression vectors can beconstructed.

These vectors may be generally administered to mammals including humansin the form of injections depending on the method of introducing intothe living body. In the case of virus vectors, they may be administeredas they are. Injections may be prepared according to a standard method,and for example after they are dissolved in a suitable solvent (a buffersuch as PBS, physiological saline, sterile water etc.), they may befilter-sterilized as desired with a filter etc. and then filled into asterile container for formulation. Commonly used carriers may be addedto the injections, as necessary. They may also be in the form ofliposome formulations described below.

In order to use the expression vectors thus obtained for the treatmentof allergic diseases, they can be administered in a standard method.Specifically, such methods include, for example, the lipofection method,the phosphate-calcium coprecipitation method, the DEAE-dextran method,the electroporation method, direct DNA injection methods using microglass capillaries, and the like. Furthermore, there are methods ofintroducing genes into the tissue. Such methods include, for example,gene introduction with internal type liposomes, gene introduction withelectrostatic type liposomes, HVJ-liposome methods, improvedHVJ-liposome methods (HVJ-AVE liposome methods), receptor-mediated geneintroduction, methods of introducing DNA molecules into the celltogether with carriers (metal particles) by a particle gun, in vivoelectroporation, and the like. There can also be used a method ofdissolving a non-virus vector expression plasmid into physiologicalsaline and using it as it is (the so-called naked-DNA directintroduction), a method of introduction with positively chargedpolymers, and the like.

When the expression vector is a virus vector, it can be used as it is,or can be administered in the form of an injection as described above.

Expression vectors containing the gene encoding α antigen, an analogthereof or a mutant thereof are usually administered to the skin,muscles, the abdominal cavity etc. of mammals including humans. Thedosage may vary depending on the type of the expression vector, dosageform, dosage regimen, the subject patient, type of disease etc., and isgenerally, as the expression vector, about 0.005 to about 2 mg,preferably about 0.1 mg to about 1 mg, generally once daily over severalmonths for a total of a few times.

In accordance with the present invention, when Mycobacterium-derived αantigen protein, an analog protein thereof, or a mutant protein thereofis used as it is, it may generally be administered pareterally, forexample intravenously, intramuscularly, intraperitoneally,subcutaneously, topically, etc. When administered parenterally, it maybe given in the form of an injection, local application form, etc.

As injections, there can be mentioned sterile solutions, suspensions, orthe like. As topical applications, there can be mentioned creams,ointments, lotions, sprays, aerosols, transdermal formulations (commonpatches, matrices etc.), and the like. These formulations may beprepared by conventional methods in combination with pharmaceuticallyacceptable excipients, additives etc. As pharmaceutically acceptableexcipients and additives, there can be mentioned carriers, binders,flagrants, buffers, thickeners, colorants, stabilizers, emulsifyingagents, dispersants, suspending agents, preservatives, and the like.

Specifically, as injections, there can be mentioned solutions,suspensions, and emulsions and the like. For example, the α antigenprotein may be added into a PBS buffer, physiological saline, sterilewater etc., to which albumin may be added as desired, which isfilter-sterilized with a filter etc. and then filled into sterilecontainers for formulation. They may also be lyophilized and dissolvedto prepare injections at the time of administration. Ointments andcreams used as topical applications may be formulated by adding the αantigen protein together with a thickener or a gelling agent to anaqueous or oily base. As the base, there can be mentioned for examplewater, liquid paraffin, plant oils (peanut oil, castor oil, etc.) andthe like. As thickeners, there can be mentioned for example softparaffin, aluminum stearate, cetostearyl alcohol, polyethylene glycol,lanoline, hydrogenated lanoline, beeswax, and the like. Lotions may beformulated in a standard method by adding one or more ofpharmaceutically acceptable stabilizers, suspending agents, emulsifyingagents, dispersants, thickeners, colorants, flagrants etc. to an aqueousor oily base. Sprays, aerosols, patches, matrices, etc. can also beformulated according to standard methods. These local applications maycontain, as desired, preservatives such as methyl hydroxybenzoate,propyl hydroxybenzoate, chloro cresol, and benzalkonium chloride, andbactericidal agents.

The dosage and the number of administration of the α antigen protein, aderivative thereof, or a mutant proteins thereof may vary depending onthe symptom, age, body weight, dosage regimen, etc., and whenadministered as injections, generally about 1 mg to about 10 mg,preferably about 1 mg to about 5 mg is administered once or severaltimes in divided doses. When topically administered, generally about 10μg of the dose may be administered over several days.

The present invention will now be specifically explained with referenceto the following examples, but these examples should not be construed tolimit the present invention.

EXAMPLE 1

Effect of an Expression Vector Containing an α Antigen-Encoding Gene onAtopic Dermatitis

(1) Construction of Expression Vector

By inserting the α antigen gene (α-K) of Mycobacterium kansasiicomprising the base sequence from positions 390-1244 set forth in SEQ IDNO: 1 into the KpnI-ApaI site of pcDNA3.1 (Invitrogen CA), and thenintegrating it downstream of the CMV promoter and the TPA signalpeptide, an expression vector pcDNA-α-K having the construction shown inFIG. 1 was constructed as an active ingredient of a pharmaceuticalcomposition for the treatment of allergic diseases of the presentinvention (Infect. Immun. 58: 550-556, 1996).

(2) Administration of an Expression Vector to a Mouse Model of AtopicDermatitis

i) Method

Transgenic mice (CTg) that express skin-specific Caspase-1 and that arein the Th2-dominant immunological state were used (J. Immunol. 165:997-1003, 2000; Nat. Immunol. 1: 132-137, 2000; Proc. Nah. Acod. Sci.USA 99;11340-11345, 2002). These mice developed atopic dermatitis fromweek 8 after birth.

To CTg mice at week 4, 10 and 12 (4, 10 and 12 week old) after birth,100 μg of the expression vector constructed in the above (1) dissolvedin PBS was intraperitoneally administered. Then, for the 4, 10 and 12week old CTg mice, at week 8 after the administration, serum levels ofhistamine and IgE as well as the expression level of interleukin-4 mRNAin the skin were investigated. Levels of histamine and IgE were measuredby RIA, and interleukin-4 mRNA by RT-PCR.

ii) Result

The result obtained is shown in Table 1. As can be seen from Table 1, inthe CTg mouse group that received the expression vector, no increases inblood levels of IgE or histamine were observed, and were in the normalrange. Also, interleukin-4 mRNA in the skin became undetectable. Duringthe observation period, no development of dermatitis or no scratchbehavior were seen.

In contrast, no treatment CTg mice developed dermatitis at week 8 andexhibited scratch behavior. Also, blood levels of IgE and histamine wereelevated.

TABLE 1 Effect on atopic dermatitis Histamine (nM/l) IgE level (μg/ml)Skin IL-4 mRNA 8 weeks Littermate 8 weeks 8 weeks Time of Before afterno treatment after Before after administration administrationadministration mice administration administration administration  4 weekold 158 15 16 0 ++ − 10 week old 230 47 19 0 ++ − 12 week old 200 170 350 ++ −

In FIG. 2, photographs of various CTg mice are shown. A photograph intop left shows CTg mice at the onset of atopic dermatitis, and one intop right shows 12 week old littermate CTg mice that received notreatment. A photograph in bottom left shows a result in which alittermate CTg mice was cured by the intramuscular injection ofprednisolone for 7 days. A photograph in bottom right shows the effectof treatment on week 8 after the intraperitoneal administration of theexpression vector constructed in the above (1) to 4 week old CTg mice.After the administration of the expression vector, no development ofdiseases was seen during the observation period (one year). Thesephotographs clearly indicate that the administration of the expressionvector containing the α antigen-encoding gene can very effectivelyprevent the onset of or treat atopic dermatitis.

EXAMPLE 2

Effect of the α Antigen Protein on Atopic Dermatitis

i) Method

Mycobacterium kansasii was cultured in the Sauton medium for 3 weeks,and the culture supernatant was precipitated with 80% ammonium sulfateto prepare a protein fraction. The protein fraction was developed andseparated by two dimensional electrophoresis on the gel, and from thecorresponding spot on the gel, α antigen protein was extracted andpurified (Scand. J. Immunol. 43: 202-209, 1996; J. Bacteriol. 170:3847-3854, 1988; Hiroshima J. Med. Sci. 32: 1-8, 1983), and dissolved inPBS at a concentration of 1 mg/ml. One μl each of this α antigen proteinsolution or the control PBS solution was applied on the head of 4 weekold CTg mice (N=3 for each) once daily for one week, and then thecondition of hair was visually inspected to assess the effect.

ii) Result

When the effect was judged by the amount of remaining hair as + (hairgrowth is seen) or − (no hair growth is seen), hair on the head wasscratched away (due to itching) and the festered skin was exposed (−) inthe control to which the PBS solution was only applied, whereas in the αantigen protein-application group hair on the head remained as it was (+to ++).

EXAMPLE 3

Effect of Expression Vector Containing the α Antigen-Encoding Gene onAsthma

1) Method

BALB/c mice (N=6 for each group) were immunized with intraperitoneallyadministration of 10 μg of ovalbumin and 1 mg alum on day 0 and day 14after the start of the experiment. For five days from day 21, theanimals were allowed to inhale aerosol of 5% ovalbumin to create anasthma model. The expression vector (100 μg) constructed in Example1-(I) and the heated BCG dead organism (100 μg) were intraperitoneallyadministered in equal amounts, respectively, on day 0 and day 14. On day25 after the start of the experiment, serum levels of IgE, proteinconcentration in the alveolar lavage, and eosinophil counts weredetermined for judgment of effect, and furthermore eosinophilinfiltration in the lung tissue was examined by an eosinophil staining(Luna stain). Also the lung tissue was histologically examined.

ii) Result

Serum levels of IgE, protein concentration in the alveolar lavage,eosinophil counts, eosinophil infiltration, and the histological imageof the lung are shown in FIG. 3 to FIG. 7, respectively. As can be seenfrom the result in FIG. 3, serum levels of IgE significantly decreasedin the α antigen gene-containing expression vector administration groupcompared to the no treatment group. As can be seen from the result inFIG. 4, protein concentration in the alveolar lavage which is an indexof inflammatory reaction was apparently low in the expression vectoradministration group compared to the no treatment group and the BCGadministration group. As can be seen from the result in FIG. 5, in theexpression vector administration group, the number of eosinophils thatinfiltrate the lung lavage was apparently repressed and the effect wasmore pronounced than the BCG administration group. As can be seen fromthe result in FIG. 6, infiltration of a large number of eosinophils wasseen in the no treatment group, but not in the expression vectoradministration group. As can be seen from the result in FIG. 7, apparentallergic inflammation was noted in the no treatment group whereas theexpression vector administration group exhibited little difference fromthe normal mice.

From the foregoing, in the α antigen gene-containing expression vectoradministration group, a marked effect of suppressing asthma symptoms wasnoted close to the no treatment normal mice, and the effect wasconsidered to be greater than the administration of an equal amount ofBCG.

INDUSTRIAL APPLICABILITY

As described above, when the α antigen, an analog thereof, or anexpression vector containing the gene encoding those analogs isadministered to atopic dermatitis, histamine release is inhibited, andfurthermore the production of IgE and interleukin-4 is suppressed,improving skin diseases and thereby exhibiting a highly effective effectfor the treatment of atopic dermatitis. Furthermore, the α antigen, ananalog protein thereof or an analog protein thereof also exhibits highlyeffective effect for the treatment of atopic dermatitis. The α antigen,an analog protein thereof or an analog protein thereof is considered toexhibit the effect of improving the Th2 type cytokine-dominantimmunological state, and therefore the α antigen, an analog thereof orthe gene encoding an analog thereof, and the α antigen protein, ananalog protein thereof or a mutant protein thereof is very effective forthe prevention or treatment of atopic diseases such as atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis, andmore broadly allergic diseases.

1. A method of treating allergic disease by shifting the Th2cytokine-dominant state to a Th1 cytokine-dominant state, comprising:administering to a mammal in need thereof an effective amount of apolynucleotide encoding a Mycobacterium kansasii-derived α antigencomprising the amino acid sequence of SEQ ID NO: 2, wherein said mammalis afflicted with an allergic disease; and said allergic disease isselected from atopic dermatitis, asthma, allergic rhinitis, and allergicconjunctivitis.
 2. The method according to claim 1, wherein thepolynucleotide is present in an expression vector.
 3. The methodaccording to claim 1, wherein said mammal is human.
 4. A method ofimmunizing a healthy mammal against an allergic disease, comprisingadministering to the mammal an effective amount of a polynucleotideencoding a Mycobacterium kansasii-derived α antigen comprising the aminoacid sequence of SEQ ID NO:
 2. 5. The method according to claim 4,wherein the allergic disease is selected from the group consisting ofasthma, atopic dermatitis, allergic rhinitis, and allergicconjunctivitis.
 6. The method according to claim 4, wherein thepolynucleotide is present in an expression vector.
 7. The methodaccording to claim 4, wherein said mammal is a human.
 8. A method oftreating an allergic disease in a mammal afflicted with an allergicdisease, comprising administering to the mammal an effective amount of apolynucleotide encoding a Mycobacterium kansasii-derived α antigencomprising the amino acid sequence of SEQ ID NO:
 2. 9. The methodaccording to claim 8 wherein the polynucleotide is present in anexpression vector.
 10. The method according to claim 8, wherein theallergic disease is selected from the group consisting of allergicrhinitis and allergic conjunctivitis.
 11. The method according to claim8, wherein said mammal is human.